Prolactin increases survival and migration of ovarian cancer cells: Importance of prolactin receptor type and therapeutic potential of S179D and G129R receptor antagonists
Highlights
► Ovarian cancer cell lines express prolactin and prolactin receptors. ► A functional autocrine prolactin growth loop was demonstrated. ► Cells with high long: short prolactin receptor ratios had increased growth rates. ► Prolactin increased growth, survival and migration. ► Two prolactin receptor antagonists decreased growth, survival and migration.
Introduction
Ovarian cancer is the most lethal of the gynecological cancers. Accepted treatment usually involves surgical de-bulking of the tumor and chemotherapy, but even when this occurs 20–30% of patients never experience a period of remission and, even the majority who do, eventually relapse [1]; better treatment regimens are therefore needed. In recent years, several reports have suggested some relationship between PRL and ovarian cancer. For example, Mor et al. [2] found that serum levels of a combination of analytes allowed 95% prediction of ovarian cancer, and elevated serum PRL was one of the four key analytes; serum PRL averaged 1 ng/ml in normal controls and 40 ng/ml in patients with ovarian cancer. In another study, 15 of 17 primary human ovarian cancers were reported to express some form of PRLR, at least at the mRNA level [3].
PRLRs belong to the superfamily of cytokine single transmembrane domain receptors [4]. Upon binding of the ligand to dimerized receptors, a conformational change occurs that activates specific associated kinases [5], [6], [7], [8], [9]. Several PRLR isoforms are formed through alternative splicing, including a long form (LF) and two short forms (SF1a and SF1b) [10], [11]. These different isoforms mediate very different biological effects, with the SFs acting as dominant negatives for signaling through Stat5, but themselves activating other signaling pathways [6], [10], [11], [12], [13]. The end result of PRL signaling therefore depends on the types and ratios of receptors expressed.
In the current study, we have examined the expression of the LF and both SFs of the human PRLR in three ovarian cancer cell lines and have linked the relative expression of the LF and SF1b to the effects of PRL as a factor influencing both cell number and cell migration. In addition, we have determined that stimulatory effects of PRL on both growth and migration can be antagonized by the PRLR antagonists, G129R- and S179D-PRL. This is of greatest importance for tumors expressing autocrine PRL where dopamine agonists would be ineffectual.
Section snippets
Ligands
PRL and S179D-PRL were purified recombinant human proteins, produced in Escherichia coli as described previously [14]. S179D-PRL is human PRL in which the serine at position 179 has been replaced with an aspartate. G129R-PRL (glycine at position 129 in human PRL replaced with an arginine) was a generous gift from Dr. Wen Chen (Clemson University, SC), also produced as a recombinant protein in E. coli, as described by Zhang et al. [15].
Cell culture and transfection
Human ovarian cancer cells (TOV21G, OV90 and TOV112D,
Expression and subtypes of prolactin receptors in ovarian cancer cells
Human ovarian carcinoma cells have been reported to express the LF PRLR [3] at the mRNA level, but whether human ovarian cancer cells also expressed SFs of the PRLR was unknown. Fig. 1 shows the result of both classical and real time RT–PCR using primers specific for the LF, SF1a or SF1b receptors. Messenger RNAs for all three forms of the PRLR were detected in each of the ovarian cancer lines examined (TOV21G, OV90 and TOV112D) (Fig. 1A and C). TOV21G is derived from a clear cell adenoma and
Discussion
PRL has been demonstrated or suggested to be an autocrine growth factor in a variety of normal and cancerous tissues and in some cases greater dependence on the autocrine growth loop has been demonstrated for cancerous cells reviewed in [18]. The expression of both PRL and various forms of the PRLR in ovarian cancer cells found in our study suggested that there could be a PRL autocrine growth loop in ovarian cancer cells. This possibility was borne out by the decrease in cell number that
Conflict of interest
The authors declare no competing financial interests in relation to the studies described. AMW is the inventor on a patent held by the Regents of the University of California in regard to the use of S179D PRL as a prolactin antagonist.
Funding
This work was supported by DOD OCRP Grant OC073294 to DT and California Breast Cancer Research Program Grant 10PB-0127 to AMW.
Author contribution statement
D.T., K.E.C. and T.K. performed the experiments on a study directed by D.T. and A.M.W. Data were analyzed by D.T., K.E.C. and A.M.W. and the manuscript was written by D.T. and A.M.W.
Acknowledgement
Dr. Mary Lorenson is acknowledged for her intellectual contributions during development of the study.
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